Purpose:
Tear film hyperosmolarity is commonly observed in dry eye, but its exact role in the pathogenesis of this ocular surface disorder is unknown. As a localized form of autoimmune disease, dry eye involves disruption of the ocular surface’s immune homeostasis by still uncharacterized stimuli. The purpose of this work was to evaluate whether hyperosmolar stress could affect conjunctival immune tolerance in mice.

Methods:
8-to-12 week-old female Balb/c mice were instilled isoosmolar (0.3 Osm) or hyperosmolar (3 Osm) saline on both eyes 3 times daily for 5 days. Ovalbumin (OVA) was also instilled or OVA-pulsed dendritic cells (DCs) were subconjunctivally injected at different time points. Induced T cell responses were measured after subcutaneous immunization with OVA by the delayed-type hypersensitivity (DTH) assay. In addition, T cells in draining lymph nodes (day 5) were evaluated by flow cytometry. Supernatants from Pam212 epithelial cells exposed to iso- or hyperosmolar medium were assayed for cytokines or in a phagocytosis assay with the Raw 264.7 macrophage cell line.

Results:
Compared to non-instilled immunized mice, OVA-instilled mice developed reduced DTH responses (i.e. were tolerized), as did their OVA+0.3Osm saline-instilled cage mates (p<0.05). By contrast OVA+3Osm saline-instilled mice exhibited full DTH responses (i.e. were not tolerized). In the latter, T cells in draining lymph nodes showed increased expression of activation (CD69, CD25) and memory (CD44) markers. Local injection of immature DCs in 3Osm saline-instilled mice, but not in 0.3Osm saline-instilled mice, induced increased T cell proliferation in the draining lymph nodes. Consistently, supernatants from Pam212 cells exposed to hyperosmolar medium for 4h, but not to control medium, had higher interleukin-1β and -6 levels and increased the phagocytic activity of macrophages.

Conclusions:
Hyperosmolar stress is sufficient to abolish conjunctival immune tolerance to a harmless antigen in Balb/c mice, suggesting that this event by itself can skew the immune response at the ocular surface. The hyperosmolarity-induced proinflammatory mucosal environment favors local maturation of DCs and T cell responses in the draining lymph nodes. These results altogether suggest that hyperosmolar stress might play a role in dry eye initiation as an immune disruptor.